An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase,JBIC Journal of Biological Inorganic Chemistry

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An isoform-specific pivot modulates the electron transfer between the flavin mononucleotide and heme centers in inducible nitric oxide synthase
JBIC Journal of Biological Inorganic Chemistry ( IF 2.7 ) Pub Date : 2020-10-14 , DOI: 10.1007/s00775-020-01824-w
Huayu Zheng _{1,

2} , Jinghui Li ₁ , Changjian Feng _{1,

2}

Affiliation

Intraprotein interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme centers is an obligatory step in nitric oxide synthase (NOS) enzymes. An isoform-specific pivotal region near Leu406 in the heme domain of human inducible NOS (iNOS) was proposed to mediate the FMN-heme domain–domain alignment (J Inorg Biochem 153:186–196, 2015). The FMN-heme IET rate is a measure of the interdomain FMN/heme complex formation. In this work, the FMN-heme IET kinetics in the wild type (wt) human iNOS oxygenase/FMN (oxyFMN) construct were directly measured by laser flash photolysis with added synthetic peptide related to the pivotal region, in comparison with the wt construct alone. The IET rates were decreased by the iNOS HKL peptide in a dose-saturable fashion, and the inhibitory effect was abolished by a single L406 → E mutation in the peptide. A similar trend in change of the NO synthesis activity of wt iNOS holoenzyme by the peptides was observed. These data, along with the kinetics and modeling results for the L406T and L406F mutant oxyFMN proteins, indicated that the Leu406 residue modulates the FMN-heme IET through hydrophobic interactions. Moreover, the IET rates were analyzed for the wt iNOS oxyFMN protein in the presence of nNOS or eNOS-derived peptide related to the equivalent pivotal heme domain site. These results together indicate that the isoform-specific pivotal region at the heme domain specifically interacts with the conserved FMN domain surface, to facilitate proper interdomain docking for the FMN-heme IET in NOS.

Graphic abstract

中文翻译：

异构体特异性枢轴调节诱导型一氧化氮合酶中黄素单核苷酸和血红素中心之间的电子转移

黄素单核苷酸 (FMN) 和血红素中心之间的蛋白质内域间电子转移 (IET) 是一氧化氮合酶 (NOS) 的必需步骤。人类诱导型 NOS (iNOS) 血红素结构域中 Leu406 附近的同种型特异性关键区域被提议介导 FMN-血红素结构域-结构域比对 (J Inorg Biochem 153:186–196, 2015)。 FMN-血红素 IET 率是域间 FMN/血红素复合物形成的量度。在这项工作中，与单独的 wt 构建体相比，通过添加与关键区域相关的合成肽的激光闪光光解直接测量野生型 (wt) 人 iNOS 加氧酶/FMN (oxyFMN) 构建体中的 FMN-血红素 IET 动力学。 iNOS HKL 肽以剂量饱和方式降低 IET 率，并且肽中的单个 L406 → E 突变消除了抑制作用。观察到肽对wt iNOS 全酶NO合成活性变化的类似趋势。这些数据以及 L406T 和 L406F 突变 oxyFMN 蛋白的动力学和建模结果表明，Leu406 残基通过疏水相互作用调节 FMN-血红素 IET。此外，在与等效关键血红素结构域位点相关的 nNOS 或 eNOS 衍生肽存在的情况下，分析了 wt iNOS oxyFMN 蛋白的 IET 率。这些结果共同表明，血红素结构域的异构体特异性关键区域与保守的 FMN 结构域表面特异性相互作用，以促进 NOS 中 FMN-血红素 IET 的正确结构域间对接。

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更新日期：2020-10-14

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